PROJECT SUMMARY The long-term goals of this research program are to determine at a molecular level how long noncoding RNAs (lncRNAs) participate in chromatin-mediated gene silencing. Many lncRNAs act in the nucleus to regulate gene expression through scaffolding of chromatin regulatory machinery. The identification of lncRNAs far outpaces detailed investigations, hence the mechanisms that govern these lncRNA-mediated events are not yet well-understood. We will address four major outstanding questions in the field: 1) How do lncRNAs target specific regions of the genome? 2) Do lncRNAs require structural remodeling for activation of chromatin repression machinery? 3) How can a lncRNA contribute to halting gene transcription? 4) What is the full-extent that a lncRNA can scaffold protein interactions on chromatin? Our immediate goals are to focus on the model lncRNA HOTAIR while longer-term goals will investigate additional lncRNAs for which much less is known. HOTAIR is transcribed from one developmentally-regulated HOX gene cluster and regulates many genes in trans through the Polycomb silencing complex PRC2. Our recent progress has identified a new key player in dictating the specificity of HOTAIR function and has suggested a model where HOTAIR uses a protein matchmaker to mediate RNA-RNA base-pairing interactions with the nascent transcripts of target genes. We will approach this model using the questions framed above to uncover a new level of mechanistic detail for this lncRNA. A multi-faceted approach will be used, merging biochemical reconstitution with cutting edge proteomics and genomics, to uncover how lncRNAs use their reservoirs of RNA sequence information to target and scaffold heterochromatin formation. These studies will generate a model for lncRNA mechanism in gene regulation that may be broadly applicable to other lncRNA pathways. We will also highlight potential molecular targets to disrupt the HOTAIR activity that promotes metastasis in many cancers. Relevance to public health Long noncoding RNAs are produced from regions of the human genome originally thought to be junk DNA. Many lncRNAs participate in epigenetic mechanisms of gene regulation and mis-regulation can lead to diseases such as cancer. LncRNAs are therefore clear candidates to provide a missing link to understanding the molecular mechanisms of many human diseases for which there is a hidden heritability factor that has not yet been identified.